The aim of this project is to develop and validate a fast, robust, reproducible, and high-resolution method of capillary sieving electrophoresis with a cationic surfactant for size-separations of proteins. Development of this method will represent a quantum leap in the proteomics technology bringing a fast, reproducible and fully automatable method for separation of proteins with unparalleled separation efficiency and reproducibility. The method will be ready for integration into automatable 2- dimensional capillary electrophoresis (2-D CE) of proteins. While automated 2-DE of proteins is highly desired by many protein researchers, cumbersome, laborious, and slow 2-dimensional slab gel electrophoresis remains a golden standard for protein separations. The proposed method has a strong potential to significantly contribute to the progress of proteomic technology and make search for protein biomarkers and target discoveries more efficient. The proposed method will also significantly improve accuracy of immunoanalysis and significantly reduce the analysis time when combined with the Western blot. Last but not least, the simple one- dimensional size separation and characterization of proteins. At high end, it will allow automated high throughput qualitative and quantitative analysis of a large number of samples in an array of capillaries and also high-speed analysis on a microchip. It is likely to address needs of a high number of researchers. In Phase I, we will develop and validate a method of capillary sieving electrophoresis with a cationic surfactant for one-dimensional quantitative analysis of proteins and their molecular weight determination. The method will have unmatched analytical characteristics, analyzing proteins in less than 12 minutes, with the separation efficiency exceeding 1,000,000 plates/m, and run-to-run reproducibility of migration times below 0.3%. In Phase I we will (1) optimize the composition of the sieving matrix, (2) validate CZECH as an analytical method for quantitative analysis of proteins, (3) validate CZECH as a method for molecular weight determination of proteins;(4) develop a protocol for sample preparation of refractory proteins to normalize their migration according to their molecular weights;(5) develop a Ferguson method to determine molecular mass of refractory proteins not responding to the modified sample preparation.. The proposed method will revolutionize protein analysis and enable advanced separation technologies that are otherwise impossible or difficult to perform. PUBLIC HEALTH RELEVANCE: The aim of this project is to develop and validate a fast, robust, reproducible, and high-resolution method of capillary sieving electrophoresis with a cationic surfactant for size-separations of proteins. The method will characterize separated proteins by determining their molecular weights in the range 3,500 - 400,000. Suppression of electroosmotic flow will allow separation efficiencies over one million theoretical plates/m and run-to-run reproducibility of migration times below 0.3%. The method will allow significantly improved 2-D separations of proteins.